This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent advances in imaging mass spectrometry present need for improving techniques in terms of spatial resolution, accuracy and MS performance. With better ion and laser optics, and more mature methodologies, development of accurate sample positioning device is gaining importance. While piezoelectric devices have superb positioning accuracy (~50 nm), their range of motion is limited. "Stacked-stages" with longer travels have a tradeoff in accuracy (~5 [unreadable]m) and speed, are bulkier and hard to implement in a vacuum environment. They also produce substantial RF interference, hence unsuitable for FT-ICR mass spectrometry. This work (Aizikov et al., 2009) presents a two-dimensional, vacuum compatible sample positioning stage capable of submicron accuracy with range of motion of 100x100 mm operating at ~1x10-8 mbar. It is a collaborative work with Ron Heeren's group at FOM Institute for Atomic and Molecular Physics (AMOLF) in Amsterdam. The two-dimensional stage is a single block design consisting of three pieces, an upper and lower plate, and the central block which hosts two axis motors, linear encoders, position limit switches, and two temperature sensors. The system is controlled by a dual loop proportional (position) gain controller with an internal velocity loop effectively comprising a PID arrangement. Velocity feed forward is added to maximize the positioning speed. Software interface to the stage controller is achieved via a dll without external dependencies, which allows for either building lightweight standalone applications, or seamless integration with existing platforms. To test the capabilities of this stage as a proof of principle, a custom built FT-ICRMS imaging instrument was equipped with the device (built and operated in Ron Heeren's group). BUSM graduate student Kostya Aizkhov participated in the analyses. A model protein, Sivinase was subjected to Trypsin and CNBr cleavage, followed by LC separation of the resulting peptides coupled to online MALDI spotting. The spatial resolution of individual peptides was assessed via a fully automated MALDI imaging experiment in a microprobe mode. Eight prominent ions assignable to Sivinase peptides through the use of Mascot peptide mass fingerprinting were determined to occupy distinct and discrete spatial location on the target within 10 x 10 mm distance. Performance evaluation and experimental results show that characteristics of the device easily meet MALDI FT-ICR MS imaging requirements in terms of positioning accuracy, speed, robustness, and RF interference. It is compatible with most of the ionization sources in terms of size and vacuum requirements. Even though the stage will work in the majority of SIMS imaging experiments, further improvements in accuracy (possible through the use of lower wavelength optical linear encoders) would allow this device to be used for virtually every need of contemporary imaging mass spectrometry. A manuscript is in preparation.